Immunotherapies employing self-assembling vaccines

ABSTRACT

Provided herein are self-assembling pharmaceutical compositions comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to four biotinylated components, and further wherein at least two of the four biotinylated components are not identical.

RELATED APPLICATIONS

This application is a 371 National Stage Application of PCT/US09/041029filed on Apr. 17, 2009; which claims priority to U.S. ProvisionalApplication Ser. No. 61/046,195, filed Apr. 18, 2008, the entirecontents of which are expressly incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 16, 2011, isnamed MAA01801.txt and is 37,053 bytes in size.

BACKGROUND

Immunization with vaccines remains a cornerstone of protection againstthreat of disease and infection. The key difficulty in vaccinedevelopment is rapidly matching a vaccine, or antitoxin, to a specificthreat. Current vaccine development strategies rely on theidentification and characterization of antigens that can be targeted tosuccessfully eradicate infection or disease. Current vaccine developmentstrategies are time- and labor-intensive and can only commence once athreat emerges. Such strategies are also impractical for generatingpersonalized vaccines to combat disease for which target antigens variesamong individuals. Current vaccine development strategies are thereforeinsufficient if a new and serious threat were to emerge, for whichsufficient time were not available to identify and characterize targetantigens before such a threat could be contained. Current vaccinedevelopment strategies are also insufficient for generating personalizedvaccines for the general population.

Thus, there is a need for a technology platform for generatingpersonalized vaccines and to contain serious threats that quicklyevolve, are fast-acting, and/or highly contagious.

SUMMARY OF THE INVENTION

In one aspect, the present invention features pharmaceuticalcompositions that can be administered to a subject to induce an immuneresponse to an antigen of interest. In one embodiment, the compositionscomprise a heat shock protein fused to a biotin-binding protein. Inanother embodiment, the compositions comprise a heat shock protein fusedto a biotin-binding protein and non-covalently bound to at least one,two, three, or four biotinylated components. In certain embodiments, thebiotinylated components are the same or different molecules.Non-limiting examples of biotinylated components include biotinylatedproteins, cells, and viruses. Non-limiting examples of biotinylatedproteins include biotinylated antigens, antibodies, and costimulatorymolecules.

The compositions of the present invention can be used prophylacticallyto raise immunity against an antigen of interest, preventing theestablishment and proliferation of viruses or cells in a subjectexpressing and exhibiting the antigen of interest or presenting portionsthereof. In this manner, prevention of the establishment andproliferation of tumor or foreign cells expressing the antigen ofinterest may be achieved. The compositions provided herein can also beused therapeutically in a subject previously infected with viruses orharboring such cells to prevent further viral or cellular proliferationor to eliminate cells of the subject that proliferate, including tumorcells expressing and exhibiting the antigen of interest or presenting aportion of the antigen.

In another embodiment, compositions comprise expression vectors capableof directing the expression of heat shock protein fused to abiotin-binding protein and/or proteins to be biotinylated, as providedherein.

In yet a further embodiment, the present invention provides methods forproducing a self-assembling pharmaceutical composition, comprisingcontacting a heat shock protein fused to a biotin-binding protein withfour biotinylated components, sufficient to form a non-covalent complexof the heat shock protein and the four biotinylated components, whereinat least two of the four biotinylated components are not identical. Thepresent invention also provides methods for inducing an immune responsein a subject, comprising administering to the subject a heat shockprotein fused to a biotin-binding protein and four biotinylatedcomponents, wherein at least two of the four biotinylated components arenot identical. The present invention further provides methods forincreasing the potency of a therapeutic in a subject, comprisingadministering to the subject a heat shock protein fused to abiotin-binding protein and four biotinylated components, wherein atleast two of the four biotinylated components are not identical.

Administration of a biotinylated monoclonal antibody and a heat shockprotein fused to a biotin-binding protein may dramatically boost theimmune efficacy and potency of available monoclonal antibodies, whichwould otherwise serve as weak vaccine immunogens. Furthermore, theavailability of a monoclonal antibody or an immune serum against aspecific target antigen may alone be sufficient to produce animmunostimulatory complex that could stimulate the production ofantibodies against an antigen of interest and the stimulation ofcytotoxic T-cells. Finally, pharmaceutical compositions described hereinwould bypass the need to create scFv specific heat shock fusion proteinsfor each antigen specific scFv and or antibody.

Thus, the present invention provides distinct advantages over existingtechnologies in allowing for: 1) preparation of vaccine to unidentified,uncharacterized antigen or antigens; 2) preparation of personalizedvaccines; 3) rapid production of pharmaceutical compositions (e.g.,vaccines), which, in turn, allow for increased capacity for productionof such of pharmaceutical compositions; and 4) “supercharging” existingtherapeutics such as monoclonal antibodies. The technologies describedherein provide for the self-assembly of fixed and potent adjuvants witha variety of different antigens, peptides, or antigen targetingmolecules, including tumor antigen specific monoclonal antibodies ofsingle-chain antibodies.

Further features and advantages will be described in the detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates modification of the pET-45b(+) expression vector. A)Schematic of the insertion of the SfiI restriction site. B) Finalmultiple cloning sites of the modified pET-45b(+) vector (SEQ ID NOS6-7, respectively, in order of appearance).

FIG. 2 shows introduction of NotI and XhoI restriction sites intoMTBhsp70. Using primers overlapping the N- and C-terminals of MTBhsp70 aNotI site and an XhoI site were introduced by PCR. The resulting PCRproduct is shown in lane 1.

FIG. 3 shows digestion of the amplified MTBhsp70 with NotI and XhoIyielding 2 bands as shown on the gel picture. Sequencing analysesestablished the presence of internal NotI and SfiI sites.

FIG. 4 shows removal of NotI and SfiI sites by using PCR based sitedirected mutagenesis. Using modified primers containing the desiredmutations, two overlapping PCR products were generated. These wereextended by PCR in absence of primers followed by the addition ofspecific primers.

FIG. 5 shows introduction of the Ovalbumin peptide (residues 254-264) atthe N-terminal of MTBhsp70. We designed a synthetic linker coding forthe Ovalbumin peptide SIINFEKL (SEQ ID NO: 4)and digested it with SfiIand NotI. Similarly, we cut the pET-45b(+) MTBhsp70 plasmid with SfiIand NotI. Both components were ligated and the ligation product was usedto transform competent bacteria. Ampicilin resistant colonies werepicked and screened by sequencing.

FIG. 6 shows partial sequencing of the N-terminal region ofOva₂₅₇₋₂₆₄-MTBhsp70 (SEQ ID NO: 8).

FIG. 7 is a schematic structure of heat shock protein fusions of aself-assembling pharmaceutical composition. Depicted are heat shockproteins MTB HSP-70 and human HSP-70 each separately fused to Avidin asthe biotin-binding protein.

FIG. 8 depicts a strategy for the assembly of wild-type and monomericAvidin.

FIG. 9 illustrates an amino acid and nucleotide comparison betweenwild-type (SEQ ID NOS 12 and 11, respectively, in order of appearance)and monomeric Avidin (SEQ ID NOS 10 and 9, respectively, in order ofappearance).

FIG. 10 illustrates sequence errors corrected by PCR based mutagenesis.FIG. 10 discloses the coding nucleotide sequence as SEQ ID NO: 13, thecoded amino acid sequences as SEQ ID NOS 13-16, respectively, in orderof appearance, and the primer sequence as SEQ ID NOS 17-20,respectively, in order of appearance.

FIG. 11 illustrates monomeric/Wild-type Avidin-Linker-MTBhsp70 sequenceswith partial MTBhsp70 sequence shown. FIG. 11 discloses the “monomeric”sequence as SEQ ID NOS 22 and 21, respectively, in order of appearance,and the “wild-type” sequence as SEQ ID NOS 24 and 23, respectively, inorder of appearance.

FIG. 12 shows refolding of Avidin-Linker-MTBhsp70 proteins found ininclusion bodies.

FIG. 13 is a schematic of a multivalent self-assembly of a heat shockprotein fusion (shown as a fusion protein of MTb HSP70 and Avidin) andfour biotinylated components (shown as biotinylated peptides 1-4) toform a multivalent self-assembled vaccine.

FIG. 14 depicts the full-length protein sequences of HSP70 fromMycobacterium tuberculosis HSP70 and Mycobacterium bovus HSP70,respectively.

FIG. 15 is a depiction of a self-assembled vaccine of a heat shockfusion protein and a biotinylated component. The heat shock fusionprotein here is shown as an HSP-Avidin Fusion, and the biotinylatedcomponent is shown as a biotinylated monoclonal antibody (BiotinylatedmAb).

FIG. 16 shows a flow cytometric analysis of interferon-γ production.

FIG. 17 shows a 2-D molecular model of an MTb HSP70-Avidin fusion with 4bound monomeric HSP-avidin fusions assembled as a tetramer around 4tetramerized avidin members, as it assembles to bind biotin.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “administer” or “administering” includes any method of deliveryof a pharmaceutical composition of the present invention, includingsystemic or localized administration. “Parenteral” means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intralesional, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intra-articular, subcapsular, subarachnoid, intraspinaland intrasternal injection, oral, epidural, intranasal, and infusion.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of the foregoing. The names of the natural aminoacids are abbreviated herein in accordance with the recommendations ofIUPAC-IUB.

The term “antibody” refers to an immunoglobulin, or derivatives thereof,which maintain specific binding ability, and proteins having a bindingdomain which is homologous or largely homologous to an immunoglobulinbinding domain. The term “antibody” is intended to encompass wholeantibodies or antigen-binding fragments thereof. These proteins may bederived from natural sources, or partly or wholly syntheticallyproduced. An antibody may be monoclonal or polyclonal. The antibody maybe a member of any immunoglobulin class from any species, including anyof the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplaryembodiments, antibodies used with the methods and compositions describedherein are derivatives of the IgG class. An antibody may be anengineered or naturally occurring antibody.

The term “antibody fragment” refers to any derivative of an antibodywhich is less than full-length. In exemplary embodiments, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fc, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly produced from a gene encoding the partial antibodysequence, or it may be wholly or partially synthetically produced. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multimolecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids.

An “antigen” refers to a target of an immune response induced by acomposition described herein. An antigen may be a protein antigen and isunderstood to include an entire protein, fragment of the proteinexhibited on the surface of a virus or an infected, foreign, or tumorcell of a subject as well as peptide displayed by an infected, foreign,or tumor cell as a result of processing and presentation of the protein,for example, through the typical MHC class I or II pathways. Examples ofsuch foreign cells include bacteria, fungi, and protozoa. Examples ofbacterial antigens include Protein A (PrA), Protein G (PrG), and ProteinL (PrL).

The term “antigen binding site” refers to a region of an antibody thatspecifically binds an epitope on an antigen.

The term “biotin-binding protein” refers to a protein, whichnon-covalently binds to biotin. A biotin-binding protein may be amonomer, dimer, or tetramer, capable of forming monovalent, divalent, ortetravalent pharmaceutical compositions, respectively, as describedherein. Non-limiting examples include anti-biotin antibodies, avidin,streptavidin, and neutravidin. The avidin may comprise mature avidin, ora sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to thesequence identified by NCBI Accession No. NP_990651. The streptavidinmay comprise, for example, a sequence that is at least 80%, 85%, 90%,95%, or 99% identical to the sequence identified by of NCBI AccessionNo. AAU48617. The term “biotin-binding protein” is intended to encompasswild-type and derivatives of avidin, streptavidin, and neutravidin,which form monomers, dimers or tetramers. Examples of such derivativesare set forth below and also described in Laitinen, O. H. (2007), “BraveNew (Strept)avidins in Biotechnology,” Trends in Biotechnology 25 (6):269-277 and Nordlund, H. R. (2003), “Introduction of histidine residuesinto avidin subunit interfaces allows pH-dependent regulation ofquaternary structure and biotin binding,” FEBS Letters 555: 449-454, thecontents of both of which are expressly incorporated herein byreference.

The term “cell” when used in the context as an antigen-containingbiotinylated component is intended to encompass whole cells or portionsthereof, provided that the portions contain the antigen of interest on asurface accessible for recognition by the immune system when apharmaceutical composition comprising the biotinylated “cell” isadministered to a subject.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “costimulatory molecule” as used herein includes any moleculewhich is able to either enhance the stimulating effect of anantigen-specific primary T cell stimulant or to raise its activitybeyond the threshold level required for cellular activation, resultingin activation of naive T cells. Such a costimulatory molecule can be amembrane-resident receptor protein.

The term “effective amount” refers to that amount of a pharmaceuticalcomposition which is sufficient to effect a desired result. An effectiveamount of a pharmaceutical composition can be administered in one ormore administrations.

The term “engineered antibody” refers to a recombinant molecule thatcomprises at least an antibody fragment comprising an antigen bindingsite derived from the variable domain of the heavy chain and/or lightchain of an antibody and may optionally comprise the entire or part ofthe variable and/or constant domains of an antibody from any of the Igclasses (for example IgA, IgD, IgE, IgG, IgM and IgY). Examples ofengineered antibodies include enhanced single chain monoclonalantibodies and enhanced monoclonal antibodies. Examples of engineeredantibodies are further described in PCT/US2007/061554, the entirecontents of which are incorporated herein by reference.

The term “epitope” refers to the region of an antigen to which anantibody binds preferentially and specifically. A monoclonal antibodybinds preferentially to a single specific epitope of a molecule that canbe molecularly defined. In the present invention, multiple epitopes canbe recognized by a multispecific antibody.

A “fusion protein” refers to a hybrid protein which comprises sequencesfrom at least two different proteins. The sequences may be from proteinsof the same or of different organisms. In various embodiments, thefusion protein may comprise one or more amino acid sequences linked to afirst protein. In the case where more than one amino acid sequence isfused to a first protein, the fusion sequences may be multiple copies ofthe same sequence, or alternatively, may be different amino acidsequences. A first protein may be fused to the N-terminus, theC-terminus, or the N- and C-terminus of a second protein.

The term “Fab fragment” refers to a fragment of an antibody comprisingan antigen-binding site generated by cleavage of the antibody with theenzyme papain, which cuts at the hinge region N-terminally to theinter-H-chain disulfide bond and generates two Fab fragments from oneantibody molecule.

The term “F(ab′)₂ fragment” refers to a fragment of an antibodycontaining two antigen-binding sites, generated by cleavage of theantibody molecule with the enzyme pepsin which cuts at the hinge regionC-terminally to the inter-H-chain disulfide bond.

The term “Fc fragment” refers to the fragment of an antibody comprisingthe constant domain of its heavy chain.

The term “Fv fragment” refers to the fragment of an antibody comprisingthe variable domains of its heavy chain and light chain. “Geneconstruct” refers to a nucleic acid, such as a vector, plasmid, viralgenome or the like which includes a “coding sequence” for a polypeptideor which is otherwise transcribable to a biologically active RNA (e.g.,antisense, decoy, ribozyme, etc), may be transfected into cells, e.g. incertain embodiments mammalian cells, and may cause expression of thecoding sequence in cells transfected with the construct. The geneconstruct may include one or more regulatory elements operably linked tothe coding sequence, as well as intronic sequences, polyadenylationsites, origins of replication, marker genes, etc.

“Host cell” refers to a cell that may be transduced with a specifiedtransfer vector. The cell is optionally selected from in vitro cellssuch as those derived from cell culture, ex vivo cells, such as thosederived from an organism, and in vivo cells, such as those in anorganism. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “immunogenic” refers to the ability of a substance to elicit animmune response. An “immunogenic composition,” or “immunogen” is acomposition or substance which elicits an immune response. An “immuneresponse” refers to the reaction of a subject to the presence of anantigen, which may include at least one of the following: makingantibodies, developing immunity, developing hypersensitivity to theantigen, and developing tolerance.

The term “including” is used herein to mean “including but not limitedto”. “Including” and “including but not limited to” are usedinterchangeably.

A “linker” is art-recognized and refers to a molecule or group ofmolecules connecting two covalent moieties, such as a heat shock proteinand biotin-binding protein. The linker may be comprised of a singlelinking molecule or may comprise a linking molecule and a spacermolecule, intended to separate the linking molecule and a moiety by aspecific distance.

The term “multivalent antibody” refers to an antibody or engineeredantibody comprising more than one antigen recognition site. For example,a “bivalent” antibody has two antigen recognition sites, whereas a“tetravalent” antibody has four antigen recognition sites. The terms“monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. referto the number of different antigen recognition site specificities (asopposed to the number of antigen recognition sites) present in amultivalent antibody. For example, a “monospecific” antibody's antigenrecognition sites all bind the same epitope. A “bispecific” antibody hasat least one antigen recognition site that binds a first epitope and atleast one antigen recognition site that binds a second epitope that isdifferent from the first epitope. A “multivalent monospecific” antibodyhas multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognitionsites, some number of which bind a first epitope and some number ofwhich bind a second epitope that is different from the first epitope.

The term “multivalent” when in reference to a self-assemblingpharmaceutical composition described herein refers to a heat shockfusion protein that is non-covalently bound to more than onebiotinylated component. The term “divalent” when in reference to aself-assembling pharmaceutical composition described herein refers to aheat shock fusion protein that is non-covalently bound to twobiotinylated components. The term “tetravalent” when in reference to aself-assembling pharmaceutical composition described herein refers to aheat shock fusion protein that is non-covalently bound to fourbiotinylated components. The biotinylated components of a multivalentpharmaceutical composition may have identical or different identities.

The term “nucleic acid” refers to a polymeric form of nucleotides,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. The terms should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single-stranded(such as sense or antisense) and double-stranded polynucleotides.

A “patient” or “subject” or “host” are used interchangeably, and eachrefers to either a human or non-human animal.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose pharmaceutical compositions which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

A “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subjectpharmaceutical composition from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Unless the context clearly indicates otherwise, “protein,”“polypeptide,” and “peptide” are used interchangeably herein whenreferring to a gene expression product, e.g., an amino acid sequence asencoded by a coding sequence. A “protein” may also refer to anassociation of one or more proteins, such as an antibody. A “protein”may also refer to a protein fragment. A protein may be apost-translationally modified protein such as a glycosylated protein. By“gene expression product” is meant a molecule that is produced as aresult of transcription of an entire or part of a gene. Gene productsinclude RNA molecules transcribed from a gene, as well as proteinstranslated from such transcripts. Proteins may be naturally occurringisolated proteins or may be the product of recombinant or chemicalsynthesis. The term “protein fragment” refers to a protein in whichamino acid residues are deleted as compared to the reference proteinitself, but where the remaining amino acid sequence is usually identicalto that of the reference protein. Such deletions may occur at theamino-terminus or carboxy-terminus of the reference protein, oralternatively both. Fragments typically are at least about 5, 6, 8 or 10amino acids long, at least about 14 amino acids long, at least about 20,30, 40 or 50 amino acids long, at least about 75 amino acids long, or atleast about 100, 150, 200, 300, 500 or more amino acids long. Fragmentsof may be obtained using proteinases to fragment a larger protein, or byrecombinant methods, such as the expression of only part of aprotein-encoding nucleotide sequence (either alone or fused with anotherprotein-encoding nucleic acid sequence). In various embodiments, afragment may comprise an enzymatic activity and/or an interaction siteof the reference protein to, e.g., a cell receptor. In anotherembodiment, a fragment may have immunogenic properties. The proteins mayinclude mutations introduced at particular loci by a variety of knowntechniques, which do not adversely effect, but may enhance, their use inthe methods provided herein. A fragment can retain one or more of thebiological activities of the reference protein.

The term “self-assembling” as used herein refers to the ability of aheat shock protein fused to a biotin-binding protein to form anon-covalent complex with biotinylated component(s) as described herein.Such ability is conferred by the non-covalent association of biotin witha biotin-binding protein.

The term “single chain variable fragment” or “scFv” refers to an Fvfragment in which the heavy chain domain and the light chain domain arelinked. One or more scFv fragments may be linked to other antibodyfragments (such as the constant domain of a heavy chain or a lightchain) to form antibody constructs having one or more antigenrecognition sites.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that the extent of the diseaseis decreased or prevented. Treatment includes (but is not limited to)administration of a composition, such as a pharmaceutical composition,and may be performed either prophylactically, or subsequent to theinitiation of a pathologic event.

The term “vaccine” refers to a pharmaceutical composition that elicitsan immune response to an antigen of interest. The vaccine may alsoconfer protective immunity upon a subject.

“Vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of preferredvector is an episome, i.e., a nucleic acid capable of extra-chromosomalreplication. Preferred vectors are those capable of autonomousreplication and/or expression of nucleic acids to which they are linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of “plasmids” which refer generally to circular doublestranded DNA loops, which, in their vector form are not bound to thechromosome. In the present specification, “plasmid” and “vector” areused interchangeably as the plasmid is the most commonly used form ofvector. However, as will be appreciated by those skilled in the art, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become subsequently known inthe art.

The term “virus” when used in the context as an antigen-containingbiotinylated component is intended to encompass whole viral particles orportions thereof, provided that the portions contain the antigen ofinterest on a surface accessible for recognition by the immune systemwhen a pharmaceutical composition comprising the biotinylated “virus” isadministered to a subject.

General

Featured herein is a novel vaccine platform that combines multipleimmunologic components to generate a highly potent immune response whenadministered to a subject. Compositions and methods of linking themultiple subunits for rapid assembly of the new vaccine are alsoprovided.

The present invention is based at least in part on the discovery that aheat shock protein fusion in non-covalent association with abiotinylated component of antibody or antigen results in a compositionthat strongly stimulates cellular, in particular cell-mediatedcytolytic, responses against the non-covalently associated proteinantigen, which responses can kill cells exhibiting the antigen.

Heat Shock Protein Fusions

A “heat shock protein” is encoded by a “heat shock gene” or a stressgene, and refers a gene that is activated or otherwise detectablyupregulated due to the contact or exposure of an organism (containingthe gene) to a stressor, such as heat shock, hypoxia, glucosedeprivation, heavy metal salts, inhibitors of energy metabolism andelectron transport, and protein denaturants, or to certain benzoquinoneansamycins. Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton,Fla. (1991). “Heat shock protein” also includes homologous proteinsencoded by genes within known stress gene families, even though suchhomologous genes are not themselves induced by a stressor.

A “heat shock protein fusion” refers to a heat shock protein linked to abiotin-binding protein. For example, a heat shock protein may be C- orN-terminally joined to a biotin-binding protein to generate a heat shockprotein fusion. When administered in conjunction with a biotinylatedcomponent provided herein, a heat shock protein fusion is capable ofstimulating or enhancing humoral and/or cellular immune responses,including CD8 cytotoxic T cell (CTL) responses, to an antigen ofinterest.

For example, but not by way of limitation, heat shock proteins which maybe used according to the invention include BiP (also referred to asgrp78), Hsp10, Hsp20-30, Hsp60 hsp70, hsc70, gp96 (grp94), hsp60, hsp40,and Hsp100-200, Hsp100, Hsp90, and members of the families thereof.Especially preferred heat shock proteins are BiP, gp96, and hsp70, asexemplified below. A particular group of heat shock proteins includesHsp90, Hsp70, Hsp60, Hsp20-30, further preferably Hsp70 and Hsp60. Mostpreferred is a member of the hsp70 family.

Hsp10 examples include GroES and Cpn10. Hsp10 is typically found in E.coli and in mitochondria and chloroplasts of eukaryotic cells. Hsp10forms a seven-membered ring that associates with Hsp60 oligomers. Hsp10is also involved in protein folding.

Hsp60 examples include Hsp65 from mycobacteria. Bacterial Hsp60 is alsocommonly known as GroEL, such as the GroEL from E. coli. Hsp60 formslarge homooligomeric complexes, and appears to play a key role inprotein folding. Hsp60 homologues are present in eukaryotic mitochondriaand chloroplasts.

Hsp70 examples include Hsp72 and Hsc73 from mammalian cells, DnaK frombacteria, particularly mycobacteria such as Mycobacterium leprae,Mycobacterium tuberculosis (MTb), and Mycobacterium bovis (such asBacille-Calmette Guerin; referred to herein as Hsp71), DnaK fromEscherichia coli, yeast, and other prokaryotes, and BiP and Grp78. Hsp70is capable of specifically binding ATP as well as unfolded proteins,thereby participating in protein folding and unfolding as well as in theassembly and disassembly of protein complexes. In a preferredembodiment, the heat shock protein is or is derived from MTb HSP 70. Thefull-length protein sequences of Mycobacterium tuberculosis HSP70 andMycobacterium bovus HSP70 are depicted in FIG. 2 as SEQ ID NOs: 1 and 2,respectively. A heat shock protein fusion to be used in conjunction withthe methods described herein may comprise a sequence that is at least80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 or 2.

Hsp90 examples include HtpG in E. coli, Hsp83 and Hsc83 yeast, and Hsp90alpha, Hsp90 beta and Grp94 in humans. Hsp90 binds groups of proteins,which proteins are typically cellular regulatory molecules such assteroid hormone receptors (eg., glucocorticoid, estrogen, progesterone,and testosterone receptors), transcription factors and protein kinasesthat play a role in signal transduction mechanisms. Hsp90 proteins alsoparticipate in the formation of large, abundant protein complexes thatinclude other heat shock proteins.

Hsp100 examples include mammalian Hsp 110, yeast Hsp104, ClpA, ClpB,ClpC, ClpX and ClpY. Yeast Hsp104 and E. coli ClpA, form hexameric andE. coli ClpB, tetrameric particles whose assembly appears to requireadenine nucleotide binding. Clp protease provides a 750 kDaheterooligomer composed of ClpP (a proteolytic subunit) and of ClpA.ClpB-Y are structurally related to ClpA, although unlike ClpA they donot appear to complex with ClpP.

Hsp100-200 examples include Grp170 (for glucose-regulated protein).Grp170 resides in the lumen of the ER, in the pre-golgi compartment, andmay play a role in immunoglobulin folding and assembly.

Naturally occurring or recombinantly derived mutants of heat shockproteins may be used according to the invention. For example, but not byway of limitation, the present invention provides for the use of heatshock proteins mutated so as to facilitate their secretion from the cell(for example having mutation or deletion of an element which facilitatesendoplasmic reticulum recapture, such as KDEL (SEQ ID NO:3) or itshomologues; such mutants are described in PCT Application No.PCT/US96/13233 (WO 97/06685), which is incorporated herein byreference).

In particular embodiments, the heat shock proteins of the presentinvention are obtained from enterobacteria, mycobacteria (particularlyM. leprae, M. tuberculosis, M. vaccae, M. smegmatis and M. bovis), E.coli, yeast, Drosophila, vertebrates, avians, chickens, mammals, rats,mice, primates, or humans.

The pharmaceutical compositions provided herein may have individualamino acid residues that are modified by oxidation or reduction.Furthermore, various substitutions, deletions, or additions may be madeto the amino acid or nucleic acid sequences, the net effect of which isto retain or further enhance the increased biological activity of theheat shock protein. Due to code degeneracy, for example, there may beconsiderable variation in nucleotide sequences encoding the same aminoacid sequence. The term “heat shock protein” is intended to encompassfragments of heat shock proteins obtained from heat shock proteins,provided such fragments include the epitopes involved with enhancing theimmune response to an antigen of interest. Fragments of heat shockproteins may be obtained using proteinases, or by recombinant methods,such as the expression of only part of a stress protein-encodingnucleotide sequence (either alone or fused with another protein-encodingnucleic acid sequence). The heat shock proteins may include mutationsintroduced at particular loci by a variety of known techniques toenhance its effect on the immune system. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press (1989); Drinkwater and Klinedinst Proc. Natl. Acad.Sci. USA 83:3402-3406 (1986); Liao and Wise, Gene 88:107-111 (1990);Horwitz et al., Genome 3:112-117 (1989).

In particular embodiments, e.g., in heat shock protein fusions involvingchemical conjugates between a heat shock protein and a biotin-bindingprotein, the heat shock proteins used in the present invention areisolated heat shock proteins, which means that the heat shock proteinshave been selected and separated from the host cell in which they wereproduced. In some embodiments where the heat shock is expressedrecombinantly as a fusion of a heat shock protein fused to abiotin-binding protein, the heat shock protein fusions used in thepresent invention are isolated heat shock protein fusions, which meansthat the heat shock protein fusions have been selected and separatedfrom the host cell in which they were produced. Such isolation can becarried out as described herein and using routine methods of proteinisolation known in the art. Maniatis et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide toProtein Purification Methods Enzymology, vol. 182, Academic Press, Inc.,San Diego, Calif. (1990).

Biotinylated Components

The term “biotinylated component” as used herein, refers to abiotinylated protein, cell, or virus. Non-limiting examples ofbiotinylated proteins include biotinylated antigens, antibodies, andcostimulatory molecules. The biotinylated component is to beadministered to a subject in conjunction with a heat shock proteinfusion as described herein.

i) Antigen-Containing Biotinylated Components

In one embodiment, the biotinylated component is derived from a subject,which may be the same or a different person to whom the pharmaceuticalcompositions are to be administered. For example, a protein, cell,and/or virus to which an immune response is desired can be isolated froma subject and optionally be amplified or cloned in vitro. The protein,cell, and/or virus may then be biotinylated in vitro using methods knownin the art. The biotinylated component may then be administered inconjunction with a heat shock protein fusion described herein to theidentical subject from which the protein, cell, and/or virus wasisolated, thus allowing for the development of personalized vaccines.Alternatively, the biotinylated component may be administered inconjunction with a heat shock protein fusion described herein to adifferent subject from which the protein, cell, and/or virus wasisolated. The latter approach allows for the development of vaccines forthe general population against a disease or infectious agent whenadministered to a general population.

Both approaches provide distinct advantages over the art, namely that,the component need only be identified to the extent that allows for itscorrelation to a specific disease or infection and allows for itsisolation from the subject. Such is a novel approach for targetingantigens whose sequence may not be known or structure even identified.Thus, the present invention allows for the preparation of pharmaceuticalcompositions to induce an immune response to known or unidentified,uncharacterized antigen or antigens. Personalized vaccines provide anadditional advantage over conventional vaccines in that HLA restrictionis not problematic because the cell or protein such as an antibody forexample, is derived from the identical host that the biotinylatedcomponent is to be administered.

In place of directly linking synthetic antigen peptides to heat shockproteins to generate a vaccine, scFVs, for example, may instead beconjugated to biotin and administered in conjunction with a heat shockprotein fusion moiety described herein, thus employing a novel fusionprotein vaccine for presenting antigen to APCs in order to generate botha humoral and CD-8 response. The scFV1s can, for example, be selectedfor by their binding to an uncharacterized antigen or to a characterizedantigen by binding studies. In this example, the scFVs are to bebiotinylated and administered in conjunction with a heat shock proteinfusion moiety, for example.

In much the same way, any protein, cell, and/or virus may bebiotinylated and administered to a subject in conjunction with a heatshock protein fusion moiety described herein, such that the biotinylatedprotein, cell, and/or virus when administered in conjunction with a heatshock fusion protein described herein targets an immune response to anantigen of interest.

An example of such a cell is a tumor cell isolated from a subject, whichis biotinylated and administered in conjunction with a heat shockprotein fusion described herein. The tumor cell prior to introduction orreintroduction into a subject in the present invention is to be treatedsuch that the cell no longer reproduces and causes harm to the subjectto which it is administered. Such may be achieved by sublethallyirradiating the tumor cell before or after biotinylation. The tumor cellexpresses antigen on its surface, the identity of which may or may notbe known or characterized. When administered to a subject in conjunctionwith the heat shock protein fusion, the non-covalent complex induces animmune response to the tumor antigen. The result is a “killer T cell”response against the cell expressing antigen, thereby targeting thediseased cell types for destruction.

The tumor cell is a cell of a type of cancer to be treated or preventedby the methods of the present invention. Such cells include, but are notlimited to, for example, a human sarcoma cell or carcinoma cell, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acutelymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, or heavy chain disease cell.

Other cells that may be biotinylated and administered in conjunctionwith a heat shock protein fusion in the same fashion as described aboveinclude any cells in a subject to which a “killer T cell” response isdesired. Examples of such cells include other diseased and/or virallyinfected cells expressing antigen on their surface. As described abovefor tumor cells, these cells prior to introduction or reintroductioninto a subject in the present invention are preferably treated such thatthe cells no longer reproduce or cause harm to the subject. Such may beachieved by sublethally irradiating the cells before or afterbiotinylation. Such cells may be treated so that they are renderednon-infectious or, if a toxin-secreting cell, no longer secrete toxin.

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents. Such infectiousagents or antigens derived therefrom, that may be biotinylated andadministered in conjunction with the present invention, include, but arenot limited to, viruses, bacteria, fungi, and protozoa. The invention isnot limited to treating or preventing infectious diseases caused byintracellular pathogens but is intended to include extracellularpathogens as well. Many medically relevant microorganisms have beendescribed extensively in the literature, e.g., see C. G. A Thomas,Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entirecontents of which is hereby incorporated by reference.

In one embodiment, virus expressing antigen or viral antigen may bebiotinylated and administered in conjunction with a heat shock proteinfusion in the same fashion as described above.

Infectious viruses of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses expressing antigen. Examplesof viruses include but are not limited to: Retroviridae (e.g. humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Bimaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class I=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Retroviruses that are contemplated include both simple retroviruses andcomplex retroviruses. The simple retroviruses include the subgroups ofB-type retroviruses, C-type retroviruses and D-type retroviruses. Anexample of a B-type retrovirus is mouse mammary tumor virus (MMTV). TheC-type retroviruses include subgroups C-type group A (including Roussarcoma virus (RSV), avian leukemia virus (ALV), and avianmyeloblastosis virus (AMV)) and C-type group B (including murineleukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus(MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV),reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). TheD-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simianretrovirus type 1 (SRV-1). The complex retroviruses include thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visnavirus, feline immunodeficiency virus (FIV), and equine infectious anemiavirus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II,simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).The foamy viruses include human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV).

Examples of RNA viruses that are antigens in vertebrate animals include,but are not limited to, the following: members of the family Reoviridae,including the genus Orthoreovirus (multiple serotypes of both mammalianand avian retroviruses), the genus Orbivirus (Bluetongue virus,Eugenangee virus, Kemerovo virus, African horse sickness virus, andColorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovineor ovine rotavirus, avian rotavirus); the family Picomaviridae,including the genus Enterovirus (poliovirus, Coxsackie virus A and B,enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus,Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirusmuris, Bovine enteroviruses, Porcine enteroviruses, the genusCardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genusRhinovirus (Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); forest virus, Sindbisvirus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);the family Rhabdoviridae, including the genus Vesiculovirus (VSV),ChanBipura virus, Flanders-Hart Park virus), the genus Lyssavirus(Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses(Marburg virus and Ebola virus); the family Arenaviridae, includingLymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, andLassa virus; the family Coronaviridae, including Infectious BronchitisVirus (IBV), Mouse Hepatitis virus, Human enteric corona virus, andFeline infectious peritonitis (Feline coronavirus).

Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to: the family Poxyiridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A, B, C, D, E and ungrouped;simian adenoviruses (at least 23 serotypes), infectious caninehepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many otherspecies, the genus Aviadenovirus (Avian adenoviruses); andnon-cultivatable adenoviruses; the family Papoviridae, including thegenus Papillomavirus (Human papilloma viruses, bovine papilloma viruses,Shope rabbit papilloma virus, and various pathogenic papilloma virusesof other species), the genus Polyomavirus (polyomavirus, Simianvacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BKvirus, JC virus, and other primate polyoma viruses such as Lymphotrophicpapilloma virus); the family Parvoviridae including the genusAdeno-associated viruses, the genus Parvovirus (Feline panleukopeniavirus, bovine parvovirus, canine parvovirus, Aleutian mink diseasevirus, etc). Finally, DNA viruses may include viruses which do not fitinto the above families such as Kuru and Creutzfeldt-Jacob diseaseviruses and chronic infectious neuropathic agents.

ii) Antibody-Containing Biotinylated Components

In another embodiment, immunotherapeutic agents, such as antibodies maybe biotinylated and administered in conjunction with a heat shockprotein fusion as described herein. Natural antibodies are themselvesdimers, and thus, bivalent. If two hybridoma cells producing differentantibodies are artificially fused, some of the antibodies produced bythe hybrid hybridoma are composed of two monomers with differentspecificities. Such bispecific antibodies can also be produced bychemically conjugating two antibodies. Natural antibodies and theirbispecific derivatives are relatively large and expensive to produce.The constant domains of mouse antibodies are also a major cause of thehuman anti-mouse antibody (HAMA) response, which prevents theirextensive use as therapeutic agents. They can also give rise to unwantedeffects due to their binding of Fc-receptors. For these reasons,molecular immunologists have been concentrating on the production of themuch smaller Fab- and Fv-fragments in microorganisms. These smallerfragments are not only much easier to produce, they are also lessimmunogenic, have no effector functions, and, because of theirrelatively small size, they are better able to penetrate tissues andtumors. In the case of the Fab-fragments, the constant domains adjacentto the variable domains play a major role in stabilizing the heavy andlight chain dimer. Accordingly, while antibodies to be used inconjunction with the present methods may include full-length or nearlyfull length engineered antibodies, smaller, single domain engineeredantibodies (that may be multivalent and multispecific) may be preferred.

The Fv-fragment is much less stable, and a peptide linker may thereforebe introduced between the heavy and light chain variable domains toincrease stability. This construct is known as a single chainFv(scFv)-fragment. A disulfide bond is sometimes introduced between thetwo domains for extra stability.

Bivalent and bispecific antibodies can be constructed using onlyantibody variable domains. A fairly efficient and relatively simplemethod is to make the linker sequence between the V_(H) and V_(L)domains so short that they cannot fold over and bind one another.Reduction of the linker length to 3-12 residues prevents the monomericconfiguration of the scFv molecule and favors intermolecular V_(H)-V_(L)pairings with formation of a 60 kDa non-covalent scFv dimer “diabody”(Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90, 6444-6448). Thediabody format can also be used for generation of recombinant bispecificantibodies, which are obtained by the noncovalent association of twosingle-chain fusion products, consisting of the V_(H) domain from oneantibody connected by a short linker to the V_(L) domain of anotherantibody. Reducing the linker length still further below three residuescan result in the formation of trimers (“triabody”, about 90 kDa) ortetramers (“tetrabody”, about 120 kDa) (Le Gall et al., 1999, FEBSLetters 453, 164-168). For a review of engineered antibodies,particularly single domain fragments, see Holliger and Hudson, 2005,Nature Biotechnology, 23:1126-1136. All of such engineered antibodiesmay be conjugated to biotin and used in the methods provided herein.

Other multivalent engineered antibodies that may be used in conjunctionwith the present embodiments are described in Lu, et al., 2003, J.Immunol. Meth. 279:219-232 (di-diabodies or tetravalent bispecificantibodies); US Published Application 20050079170 (multimeric Fvmolecules or “flexibodies”), and WO99/57150 and Kipriyanov, et al.,1999, J. Mol. Biol. 293:41-56 (tandem diabodies, or “Tandabs”).

Any of the above-described multivalent engineered antibodies may bedeveloped by one of skill in the art using routine recombinant DNAtechniques, for example as described in PCT International ApplicationNo. PCT/US86/02269; European Patent Application No. 184,187; EuropeanPatent Application No. 171,496; European Patent Application No. 173,494;PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application No. 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No.5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; Beidler et al. (1988) J. Immunol.141:4053-4060; and Winter and Milstein, Nature, 349, pp. 293-99 (1991)).Preferably non-human antibodies are “humanized” by linking the non-humanantigen binding domain with a human constant domain (e.g. Cabilly etal., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.U.S.A., 81, pp. 6851-55 (1984)).

The antigen recognition sites or entire variable regions of theengineered antibodies may be derived from one or more parentalantibodies directed against any antigen of interest. The parentalantibodies can include naturally occurring antibodies, antibodiesadapted from naturally occurring antibodies, or antibodies constructedde novo using sequences of antibodies or known to be specific for anantigen of interest. Sequences that may be derived from parentalantibodies include heavy and/or light chain variable regions and/orCDRs, framework regions or other portions thereof.

Multivalent, multispecific antibodies may contain a heavy chaincomprising two or more variable regions and/or a light chain comprisingone or more variable regions wherein at least two of the variableregions recognize different epitopes on the same antigen.

Candidate antibodies to be biotinylated, may be screened for activityusing a variety of known assays. For example, screening assays todetermine binding specificity are well known and routinely practiced inthe art. For a comprehensive discussion of such assays, see Harlow etal. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y., 1988, Chapter 6.

In one embodiment, the antibodies are monoclonal antibodies and/or havein vivo therapeutic and/or prophylactic uses against cancer. In someembodiments, the antibodies can be used for treatment and/or preventionof infectious disease. Examples of therapeutic and prophylacticantibodies include, but are not limited to, ERBITUX® (Cetuximab)(ImClone System), MDX-010 (Medarex, N.J.) which is a humanizedanti-CTLA-4 antibody currently in clinic for the treatment of prostatecancer; SYNAGIS® (MedImmune, Md.) which is a humanized anti-respiratorysyncytial virus (RSV) monoclonal antibody for the treatment of patientswith RSV infection; HERCEPTIN® (Trastuzumab) (Genentech, Calif.) whichis a humanized anti-HER2 monoclonal antibody for the treatment ofpatients with metastatic breast cancer. Other examples are a humanizedanti-CD18 F(ab′)₂ (Genentech); CDP860 which is a humanized anti-CD18F(ab′)₂ (Celltech, UK); PRO542 which is an anti-HIV gp120 antibody fusedwith CD4 (Progenics/Genzyme Transgenics); Ostavir which is a human antiHepatitis B virus antibody (Protein Design Lab/Novartis); PROTOVIR™which is a humanized anti-CMV IgG1 antibody (Protein DesignLab/Novartis); MAK-195 (SEGARD) which is a murine anti-TNF-α F(ab′)₂(Knoll Pharma/BASF); IC14 which is an anti-CD14 antibody (ICOS Pharm); ahumanized anti-VEGF IgG1 antibody (Genentech); OVAREX™ which is a murineanti-CA 125 antibody (Altarex); PANOREX™ which is a murine anti-17-IAcell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImCloneSystem); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImCloneSystem); VITAXIN™ which is a humanized anti-αVβ3 integrin antibody(Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03 which is ahumanized anti CD52 IGGI antibody (Leukosite); Smart M195 which is ahumanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™which is a chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech,Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody(Immunomedics); Smart ID10 which is a humanized anti-HLA antibody(Protein Design Lab); ONCOLYM™ (Lym-1) is a radiolabelled murineanti-HLA DIAGNOSTIC REAGENT antibody (Techniclone); ABX-IL8 is a humananti-IL8 antibody (Abgenix); anti-CD11a is a humanized IgG1 antibody(Genentech/Xoma); ICM3 is a humanized anti-ICAM 3 antibody (ICOS Pharm);IDEC-114 is a primatized anti-CD80 antibody (IDEC Pharm/Mitsubishi);ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/ScheringAG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatizedanti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanizedanti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complementfactor 5 (C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-αantibody (CAT/BASF); CDP870 is a humanized anti-TNF-α Fab fragment(Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody(Celltech); LDP-02 is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgGantibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD 40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering);SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies,respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tan-ox Biosystems); ABX-CBL is amurine anti CD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgGantibody (Medimmune/Bio Transplant); Orthoclone/OKT3 is a murineanti-CD3 IgG2a antibody (ortho Biotech); SIMULECT™ is a chimericanti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanizedanti-β2-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine antiCD18 F(ab′)₂ (Pasteur-Merieux/Immunotech-); CAT-152 is a humananti-TGF-β2 antibody (Cambridge Ab Tech); and Corsevin M is a chimericanti-Factor VII antibody (Centocor).

Self-Assembling Vaccines

Multiple biotinylated components may be administered in conjunction witha heat shock protein fusion as further described. In this way,multivalent pharmaceutical compositions may be generated andadministered to a subject. The generation of multivalent pharmaceuticalcompositions allow for the production of “supercharged,” or more potentvaccines and therapeutics. When the biotinylated component comprises anantibody, such vaccine exhibits activity improvement for marketedantibodies.

Wherein the pharmaceutical composition is multivalent, the biotinylatedcomponents to be administered may be any combination of biotinylatedcomponents described herein. For example, biotinylated components of thesame or different identities may be administered in conjunction with aheat shock protein fusion as provided herein, provided that thebiotin-binding protein, and in turn the heat shock protein fusion, ismultivalent, or capable of binding multiple biotinylated components. Asan example, the wild-type biotin-binding protein avidin has fourbiotin-binding sites and is therefore capable of binding fourbiotinylated components. In this example, the four sites are to be boundby four biotinylated components, and the biotin-binding components maybe mixed and matched based on identity in any possible permutation ofone, two, three, or four identical biotinylated components describedherein. Four identical biotinylated components may be bound to the fourbiotin-binding sites.

Therefore, an effective amount of a biotinylated component with a firstidentity may be may be administered to a subject in conjunction with aheat shock protein fused to a biotin-binding protein, sufficient to forma pharmaceutical composition comprising four parts biotinylatedcomponent of a first identity and one part heat shock protein fused to abiotin-binding protein. Alternatively, an effective amount ofbiotinylated components with a first and second identity may be may beadministered to a subject in conjunction with a heat shock protein fusedto a biotin-binding protein, sufficient to form a pharmaceuticalcomposition comprising three parts biotinylated component of a firstidentity, one part biotinylated component of a second identity, and onepart heat shock protein fusion. In another embodiment, an effectiveamount of biotinylated components with a first and second identity maybe administered to a subject in conjunction with a heat shock proteinfused to a biotin-binding protein, sufficient to form a pharmaceuticalcomposition comprising two parts biotinylated component of a firstidentity, two parts biotinylated component of a second identity, and onepart heat shock protein fusion.

Wherein the self-assembling pharmaceutical composition is divalent, aneffective amount of biotinylated component of a first identity be may beadministered to a subject in conjunction with a heat shock protein fusedto a biotin-binding protein, sufficient to form a pharmaceuticalcomposition comprising two parts biotinylated component of a firstidentity and one part heat shock protein fusion. Alternatively, aneffective amount of biotinylated components with a first and secondidentity may be may be administered to a subject in conjunction with aheat shock protein fused to a biotin-binding protein, sufficient to forma pharmaceutical composition comprising one part biotinylated componentof a first identity, one part biotinylated component of a secondidentity, and one part heat shock protein fusion.

A biotinylated component of a multivalent pharmaceutical composition mayinclude a costimulatory molecule, or a blocking group (i.e., biotinalone or biotin conjugated to a non-functional molecule). Examples ofcostimulatory molecules that may be administered in conjunction with thepresent invention include B7 molecules, including B7-1 (CD80) and B7-2(CD86), CD28, CD58, LFA-3, CD40, B7-H3, CD137 (4-1BB), and interleukins(e.g., IL-1, IL-2, or IL-12). As an example, one part biotinylatedcomponent comprising a costimulatory molecule may be administered inconjunction with i) three parts of another biotinylated componentcomprising a protein, cell or virus; and ii) one part heat shock proteinfused to a biotin-binding protein. In another example, two partsbiotinylated component comprising a costimulatory molecule may beadministered in conjunction with i) two parts of another biotinylatedcomponent comprising a protein, cell, or virus; and ii) one part heatshock protein fused to a biotin-binding protein. In another example,three parts biotinylated component comprising a costimulatory moleculemay be administered in conjunction with i) one part of anotherbiotinylated component comprising a protein, cell, or virus; and ii) onepart heat shock protein fused to a biotin-binding protein.

A pH-sensitive mutant of avidin, streptavidin, or neutravidin, forexample, may be employed to control the noncovalent interaction ofavidin-, streptavidin-, or neutravidin- to biotin, and thereby achievethe desired stoichiometry of heat shock protein fusion with the variouspermutations and combinations of biotinylated component, as describedherein. The choice of wild-type or a particular mutant form ofbiotin-binding protein such as avidin may be employed to control thedesired valency of the pharmaceutical composition (e.g., monomeric,dimeric, or tetrameric form of avidin). Monovalent or divalent vaccinesmay be similarly produced by employing heat shock fusion proteinscomprising other avidin, streptavidin, or neutravidin mutant proteinsthat bind biotin but in a monovalent or divalent fashion. An example ofan avidin mutant is described in the Exemplification section below. Anexample of a pH-sensitive point mutant of Avidin which conferspH-adjustable biotin binding is Y33H. Another mutant has substitutionsof histidine for Met96, Val115, and Ile117, optionally with histidinereplacement at Trp110. Such approaches for controllingbiotin-streptavidin binding are described in Laitinen, O. H. (2007),“Brave New (Strept)avidins in Biotechnology,” Trends in Biotechnology 25(6): 269-277 and Nordlund, H. R. (2003), “Introduction of histidineresidues into avidin subunit interfaces allows pH-dependent regulationof quaternary structure and biotin binding,” FEBS Letters 555: 449-454,the contents of both of which are incorporated herein by reference.

Methods of Producing the Self-Assembling Pharmaceutical Compositions

In one embodiment of the present invention, compositions are comprisedof two moieties: a heat shock protein fused to a biotin-binding proteinand a biotinylated component which targets the immune response to theantigen to which the immune response is desired. The present inventionprovides for fast, easy production of large amounts pharmaceuticalcomposition (e.g., vaccine) because the production of biotinylatedantigens or antibodies is well known and rapid, which, in turn, allowsfor an increased capacity for vaccine production. Because a heat shockprotein fusion of a single identity may be administered in conjunctionwith any of a number of various biotinylated components as describedherein, the heat shock fusion protein need not be synthesized de novoeach time a new target antigen of interest is identified. Therefore,such methods of production are particularly rapid once the heat shockprotein fusion to be administered is established and has been produced.

Provided are methods for making the heat shock protein fused to abiotin-binding protein. The heat shock protein may be prepared, usingstandard techniques, from natural sources, for example as described inFlynn et al., Science 245:385-390 (1989), or using recombinanttechniques such as expression of a heat shock encoding gene construct ina suitable host cell such as a bacterial, yeast or mammalian cell. Afusion protein including the heat shock protein and biotin-bindingprotein can be produced by recombinant means. For example, a nucleicacid encoding the heat shock protein can be joined to either end of anucleic acid sequence encoding the biotin-binding protein such that thetwo protein-coding sequences are sharing a common translational readingframe and can be expressed as a fusion protein including thebiotin-binding protein and the heat shock protein. The combined sequenceis inserted into a suitable vector chosen based on the expressionfeatures desired and the nature of the host cell. In the examplesprovided hereinafter, the nucleic acid sequences are assembled in avector suitable for protein expression in the bacterium E. coli.Following expression in the chosen host cell, the fusion protein can bepurified by routine biochemical separation techniques or byimmunoaffinity methods using an antibody to one or the other part of thefusion protein. Alternatively, the selected vector can add a tag to thefusion protein sequence, e.g., an oligohistidine tag as described in theexamples presented hereinafter, permitting expression of a tagged fusionprotein that can be purified by affinity methods using an antibody orother material having an appropriately high affinity for the tag.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory Press (1989); Deutscher, M. Guide to ProteinPurification Methods Enzymology, vol. 182. Academic Press, Inc. SanDiego, Calif. (1990). If a vector suitable for expression in mammaliancells is used. e.g., one of the vectors discussed below, the heat shockprotein fusion can be expressed and purified from mammalian cells.Alternatively, the mammalian expression vector (including fusionprotein-coding sequences) can be administered to a subject to directexpression of heat shock protein fusion protein in the subject's cells.A nucleic acid encoding a heat shock protein can also be producedchemically and then inserted into a suitable vector for fusion proteinproduction and purification or administration to a subject. Finally, afusion protein can also be prepared chemically.

Techniques for making fusion genes are well known in the art.Essentially, the joining of various DNA fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene may be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments may be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which may subsequently be annealed to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al., John Wiley & Sons: 1992). Accordingly, provided is anisolated nucleic acid comprising a fusion gene of a gene encoding a heatshock protein fused to a gene encoding a biotin-binding protein.

The nucleic acid may be provided in a vector comprising a nucleotidesequence encoding the heat shock protein fusion, and operably linked toat least one regulatory sequence. It should be understood that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed and/or the type of protein desired tobe expressed. The vector's copy number, the ability to control that copynumber and the expression of any other protein encoded by the vector,such as antibiotic markers, should be considered. Such vectors may beadministered in any biologically effective carrier, e.g., anyformulation or composition capable of effectively transfecting cellseither ex vivo or in vivo with genetic material encoding a chimericpolypeptide. Approaches include insertion of the nucleic acid in viralvectors including recombinant retroviruses, adenoviruses,adeno-associated viruses, human immunodeficiency viruses, and herpessimplex viruses-1, or recombinant bacterial or eukaryotic plasmids.Viral vectors may be used to transfect cells directly; plasmid DNA maybe delivered alone with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g., antibody conjugated), polylysineconjugates, gramicidin S, artificial viral envelopes or other suchintracellular carriers. Nucleic acids may also be directly injected.Alternatively, calcium phosphate precipitation may be carried out tofacilitate entry of a nucleic acid into a cell.

The subject nucleic acids may be used to cause expression andover-expression of a heat shock protein fusion protein in cellspropagated in culture, e.g. to produce fusion proteins.

Provided also is a host cell transfected with a recombinant gene inorder to express the heat shock protein fusion. The host cell may be anyprokaryotic or eukaryotic cell. For example, a heat shock protein fusionmay be expressed in bacterial cells, such as E. coli, insect cells(baculovirus), yeast, insect, plant, or mammalian cells. In thoseinstances when the host cell is human, it may or may not be in a livesubject. Other suitable host cells are known to those skilled in theart. Additionally, the host cell may be supplemented with tRNA moleculesnot typically found in the host so as to optimize expression of thepolypeptide. Other methods suitable for maximizing expression of thefusion polypeptide will be known to those in the art.

A cell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. A fusion polypeptidemay be secreted and isolated from a mixture of cells and mediumcomprising the polypeptide. Alternatively, a fusion polypeptide may beretained cytoplasmically and the cells harvested, lysed and the proteinisolated. A fusion polypeptide may be isolated from cell culture medium,host cells, or both using techniques known in the art for purifyingproteins, including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for particular epitopes of afusion.

Thus, a nucleotide sequence encoding all or part of the heat shockprotein fusion may be used to produce a recombinant form of a proteinvia microbial or eukaryotic cellular processes. Ligating the sequenceinto a polynucleotide construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial cells), arestandard procedures. Similar procedures, or modifications thereof, maybe employed to prepare recombinant fusion polypeptides by microbialmeans or tissue-culture technology in accord with the subject invention.

Expression vehicles for production of a recombinant protein includeplasmids and other vectors. For instance, suitable vectors for theexpression of a fusion polypeptide include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

In another embodiment, the nucleic acid encoding the heat protein fusionpolypeptide is operably linked to a bacterial promoter, e.g., theanaerobic E. coli, NirB promoter or the E. coli lipoprotein llppromoter, described, e.g., in Inouye et al. (1985) Nucl. Acids Res.13:3101; Salmonella pagC promoter (Miller et al., supra), Shigella entpromoter (Schmitt and Payne, J. Bacteriol. 173:816 (1991)), the tetpromoter on Tn10 (Miller et al., supra), or the ctx promoter of Vibriocholera. Any other promoter can be used. The bacterial promoter can be aconstitutive promoter or an inducible promoter. An exemplary induciblepromoter is a promoter which is inducible by iron or in iron-limitingconditions. In fact, some bacteria, e.g., intracellular organisms, arebelieved to encounter iron-limiting conditions in the host cytoplasm.Examples of iron-regulated promoters of FepA and TonB are known in theart and are described, e.g., in the following references: Headley, V. etal. (1997) Infection & Immunity 65:818; Ochsner, U. A. et al. (1995)Journal of Bacteriology 177:7194; Hunt, M. D. et al. (1994) Journal ofBacteriology 176:3944; Svinarich, D. M. and S. Palchaudhuri. (1992)Journal of Diarrhoeal Diseases Research 10:139; Prince, R. W. et al.(1991) Molecular Microbiology 5:2823; Goldberg, M. B. et al. (1990)Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journalof Bacteriology 169:2624; and Hantke, K. (1981) Molecular & GeneralGenetics 182:288.

A plasmid preferably comprises sequences required for appropriatetranscription of the nucleic acid in bacteria, e.g., a transcriptiontermination signal. The vector can further comprise sequences encodingfactors allowing for the selection of bacteria comprising the nucleicacid of interest, e.g., gene encoding a protein providing resistance toan antibiotic, sequences required for the amplification of the nucleicacid, e.g., a bacterial origin of replication.

In another embodiment, a signal peptide sequence is added to theconstruct, such that the fusion polypeptide is secreted from cells. Suchsignal peptides are well known in the art.

In one embodiment, the powerful phage T5 promoter, that is recognized byE. coli RNA polymerase is used together with a lac operator repressionmodule to provide tightly regulated, high level expression orrecombinant proteins in E. coli. In this system, protein expression isblocked in the presence of high levels of lac repressor.

In one embodiment, the DNA is operably linked to a first promoter andthe bacterium further comprises a second DNA encoding a first polymerasewhich is capable of mediating transcription from the first promoter,wherein the DNA encoding the first polymerase is operably linked to asecond promoter. In a preferred embodiment, the second promoter is abacterial promoter, such as those delineated above. In an even morepreferred embodiment, the polymerase is a bacteriophage polymerase,e.g., SP6, T3, or T7 polymerase and the first promoter is abacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.Plasmids comprising bacteriophage promoters and plasmids encodingbacteriophage polymerases can be obtained commercially, e.g., fromPromega Corp.(Madison, Wis.) and InVitrogen (San Diego, Calif.), or canbe obtained directly from the bacteriophage using standard recombinantDNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning:A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophagepolymerases and promoters are further described, e.g., in the followingreferences: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al.(1994) PNAS USA 91:4034; Dubendorff, J. W. and F. W. Studier (1991)Journal of Molecular Biology 219:45; Bujarski, J. J. and P. Kaesberg(1987) Nucleic Acids Research 15:1337; and Studier, F. W. et al. (1990)Methods in Enzymology 185:60). Such plasmids can further be modifiedaccording to the specific embodiment of the heat shock protein fusion tobe expressed.

In another embodiment, the bacterium further comprises a DNA encoding asecond polymerase which is capable of mediating transcription from thesecond promoter, wherein the DNA encoding the second polymerase isoperably linked to a third promoter. The third promoter may be abacterial promoter. However, more than two different polymerases andpromoters could be introduced in a bacterium to obtain high levels oftranscription. The use of one or more polymerases for mediatingtranscription in the bacterium can provide a significant increase in theamount of polypeptide in the bacterium relative to a bacterium in whichthe DNA is directly under the control of a bacterial promoter. Theselection of the system to adopt will vary depending on the specificuse, e.g., on the amount of protein that one desires to produce.

Generally, a nucleic acid encoding a fusion protein is introduced into ahost cell, such as by transfection, and the host cell is cultured underconditions allowing expression of the fusion protein. Methods ofintroducing nucleic acids into prokaryotic and eukaryotic cells are wellknown in the art. Suitable media for mammalian and prokaryotic host cellculture are well known in the art. Generally, the nucleic acid encodingthe subject fusion protein is under the control of an induciblepromoter, which is induced once the host cells comprising the nucleicacid have divided a certain number of times. For example, where anucleic acid is under the control of a beta-galactose operator andrepressor, isopropyl beta-D-thiogalactopyranoside (IPTG) is added to theculture when the bacterial host cells have attained a density of aboutOD₆₀₀ 0.45-0.60. The culture is then grown for some more time to givethe host cell the time to synthesize the protein. Cultures are thentypically frozen and may be stored frozen for some time, prior toisolation and purification of the protein.

When using a prokaryotic host cell, the host cell may include a plasmidwhich expresses an internal T7 lysozyme, e.g., expressed from plasmidpLysSL (see Examples). Lysis of such host cells liberates the lysozymewhich then degrades the bacterial membrane.

Other sequences that may be included in a vector for expression inbacterial or other prokaryotic cells include a synthetic ribosomalbinding site; strong transcriptional terminators, e.g., t₀ from phagelambda and t₄ from the rrnB operon in E. coli, to prevent read throughtranscription and ensure stability of the expressed protein; an originof replication, e.g., ColE1; and beta-lactamase gene, conferringampicillin resistance.

Other host cells include prokaryotic host cells. Even more preferredhost cells are bacteria, e.g., E. coli. Other bacteria that can be usedinclude Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp.,Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp.,Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp.,Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp.,Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp.,Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp.Most of these bacteria can be obtained from the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209).

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al., (1983)in Experimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83). These vectors may replicate in E. coli due the presenceof the pBR322 ori, and in S. cerevisiae due to the replicationdeterminant of the yeast 2 micron plasmid. In addition, drug resistancemarkers such as ampicillin may be used.

In certain embodiments, mammalian expression vectors contain bothprokaryotic sequences to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and17. In some instances, it may be desirable to express the recombinantprotein by the use of a baculovirus expression system. Examples of suchbaculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal comprising pBlueBacIII).

In another variation, protein production may be achieved using in vitrotranslation systems. In vitro translation systems are, generally, atranslation system which is a cell-free extract comprising at least theminimum elements necessary for translation of an RNA molecule into aprotein. An in vitro translation system typically comprises at leastribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexesinvolved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex,comprising the cap-binding protein (CBP) and eukaryotic initiationfactor 4F (eIF4F). A variety of in vitro translation systems are wellknown in the art and include commercially available kits. Examples of invitro translation systems include eukaryotic lysates, such as rabbitreticulocyte lysates, rabbit oocyte lysates, human cell lysates, insectcell lysates and wheat germ extracts. Lysates are commercially availablefrom manufacturers such as Promega Corp., Madison, Wis.; Stratagene, LaJolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, GrandIsland, N.Y. In vitro translation systems typically comprisemacromolecules, such as enzymes, translation, initiation and elongationfactors, chemical reagents, and ribosomes. In addition, an in vitrotranscription system may be used. Such systems typically comprise atleast an RNA polymerase holoenzyme, ribonucleotides and any necessarytranscription initiation, elongation and termination factors. An RNAnucleotide for in vitro translation may be produced using methods knownin the art. In vitro transcription and translation may be coupled in aone-pot reaction to produce proteins from one or more isolated DNAs.

When expression of a carboxy terminal fragment of a protein is desired,i.e. a truncation mutant, it may be necessary to add a start codon (ATG)to the oligonucleotide fragment comprising the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position may be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., (1987) PNAS USA 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, may beachieved either in vivo by expressing such recombinant proteins in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al.).

In cases where plant expression vectors are used, the expression a heatshock protein fusion may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al., 1984, Nature, 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-311) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al.,1984, Science, 224:838-843); or heat shock promoters, e.g., soybean Hsp17.5-E or Hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol., 6:559-565)may be used. These constructs can be introduced into plant cells usingTi plasmids, Ri plasmids, plant virus vectors; direct DNAtransformation; microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, New York, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.

An alternative expression system which can be used to express a proteintag or fusion protein comprising a protein tag is an insect system. Inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The PGHS-2 sequence may be cloned intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of the coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted gene isexpressed. (e.g., see Smith et al., 1983, J. Viol., 46:584, Smith, U.S.Pat. No. 4,215,051).

In a specific embodiment of an insect system, the DNA encoding the heatshock protein fusion protein is cloned into the pBlueBacIII recombinanttransfer vector (Invitrogen, San Diego, Calif.) downstream of thepolyhedrin promoter and transfected into Sf9 insect cells (derived fromSpodoptera frugiperda ovarian cells, available from Invitrogen, SanDiego, Calif.) to generate recombinant virus. After plaque purificationof the recombinant virus high-titer viral stocks are prepared that inturn would be used to infect Sf9 or High Five™ (BTI-TN-5B1-4 cellsderived from Trichoplusia ni egg cell homogenates; available fromInvitrogen, San Diego, Calif.) insect cells, to produce large quantitiesof appropriately post-translationally modified subject protein.

In other embodiments, the heat shock protein fusion and biotin-bindingprotein are produced separately and then linked, e.g. covalently linked,to each other. For example, a heat shock protein fusion andbiotin-binding protein are produced separately in vitro, purified, andmixed together under conditions under which the tag will be able to belinked to the protein of interest. For example, the heat shock proteinand/or the biotin-binding protein can be obtained (isolated) from asource in which it is known to occur, can be produced and harvested fromcell cultures, can be produced by cloning and expressing a gene encodingthe desired heat shock protein fusion, or can be synthesized chemically.Furthermore, a nucleic acid sequence encoding the desired heat shockprotein fusion can be synthesized chemically. Such mixtures ofconjugated proteins may have properties different from single fusionproteins.

Linkers (also known as “linker molecules” or “cross-linkers”) may beused to conjugate a heat shock protein and biotin-binding protein.Linkers include chemicals able to react with a defined chemical group ofseveral, usually two, molecules and thus conjugate them. The majority ofknown cross-linkers react with amine, carboxyl, and sulfhydryl groups.The choice of target chemical group is crucial if the group may beinvolved in the biological activity of the proteins to be conjugated.For example, maleimides, which react with sulfhydryl groups, mayinactivate Cys-comprising proteins that require the Cys to bind to atarget. Linkers may be homofunctional (comprising reactive groups of thesame type), heterofunctional (comprising different reactive groups), orphotoreactive (comprising groups that become reactive on illumination).

Linker molecules may be responsible for different properties of theconjugated compositions. The length of the linker should be consideredin light of molecular flexibility during the conjugation step, and theavailability of the conjugated molecule for its target (cell surfacemolecules and the like.) Longer linkers may thus improve the biologicalactivity of the compositions of the present invention, as well as theease of preparation of them. The geometry of the linker may be used toorient a molecule for optimal reaction with a target. A linker withflexible geometry may allow the cross-linked proteins toconformationally adapt as they bind other proteins. The nature of thelinker may be altered for other various purposes. For example, thearyl-structure of MBuS was found less immunogenic than the aromaticspacer of MBS. Furthermore, the hydrophobicity and functionality of thelinker molecules may be controlled by the physical properties ofcomponent molecules. For example, the hydrophobicity of a polymericlinker may be controlled by the order of monomeric units along thepolymer, e.g. a block polymer in which there is a block of hydrophobicmonomers interspersed with a block of hydrophilic monomers.

The chemistry of preparing and utilizing a wide variety of molecularlinkers is well-known in the art and many pre-made linkers for use inconjugating molecules are commercially available from vendors such asPierce Chemical Co., Roche Molecular Biochemicals, United StatesBiological, and the like.

The prepared and/or isolated heat shock protein fused to abiotin-binding protein is to be administered to a subject in conjunctionwith the desired biotinylated components, sufficient to form anon-covalent association of the biotin moiety with the biotin-bindingprotein. The heat shock protein fusion and the biotinylated component orcomponents may be administered simultaneously or sequentially. Ifadministered simultaneously, the heat shock protein fusion and thebiotinylated component or components may be administered as a mixture oras a noncovalent complex. If administered as a noncovelent complex, aheat shock protein fused to a biotin-binding protein may benoncovalently bound to the desired biotinylated components either invitro or in vivo once prepared and/or isolated.

The noncovalent complex may be produced by contacting the heat shockprotein fused to a biotin-binding protein with the biotinylatedcomponents, under conditions sufficient to promote the binding of thebiotin-binding protein with biotin, which conditions are known in theart.

Genes for various heat shock proteins have been cloned and sequenced,and which may be used to obtain a heat shock protein fusion, including,but not limited to, gp96 (human: Genebank Accession No. X15187; Maki etal., Proc. Natl. Acad. Sci. U.S.A. 87:5658-5562 (1990); mouse: GenebankAccession No. M16370; Srivastava et al., Proc. Natl. Acad. Sci. U.S.A.84:3807-3811 (1987)), BiP (mouse: Genebank Accession No. U16277; Haas etal., Proc. Natl. Acad. Sci. U.S.A. 85:2250-2254 (1988); human: GenebankAccession No. M19645; Ting et al., DNA 7:275-286 (1988)), hsp70 (mouse:Genebank Accession No. M35021; Hunt et al., Gene 87:199-204 (1990);human: Genebank Accession No. M24743; Hunt et al, Proc. Natl. Acad. Sci.U.S.A. 82:6455-6489 (1995)), and hsp40 (human: Genebank Accession No.D49547; Ohtsuka K., Biochem. Biophys. Res. Commun. 197:235-240 (1993)).

The heat shock protein fused to a biotin-binding protein may benon-covalently bound to the biotinylated component.

The component to be administered in conjunction with the heat shockprotein comprising the protein, cell, or virus may be conjugated tobiotin by means such as is known in the art. Prior to conjugation tobiotin, the protein, cell, or virus may be produced and/or isolatedusing methods known in the art. Recombinant techniques may be employedin much the same way as described herein for the heat shock proteinfusion. Once the component is produced and/or isolated, a biotinmolecule or molecules may be conjugated directly to a protein, cell, orvirus. Biotin may also be conjugated indirectly through a linker to saidprotein, cell, or virus. Biotin is to be conjugated to a region thatsterically allows for the interaction of biotin with the biotin-bindingprotein. Biotinylation kits and reagents may be purchased from Pierce(Rockford, Ill.) and used to generate the biotinylated componentsdescribed herein.

The sequences of many different antigens can be cloned and characterizedby DNA sequence analysis and included in the compositions providedherein. Bacterial vectors containing complete or partial cellular orviral genomes or antigens may be obtained from various sourcesincluding, for example, the American Tissue Culture Collection (ATCC).Additional antigens which may be used can be isolated and typed by themethods previously established for this purpose, which methods are wellknown in the art.

Methods of Using the Heat Shock Protein Fusion and BiotinylatedComponents

The heat shock protein fusion and biotinylated components describedherein can be administered to a subject to induce or enhance thatsubject's immune response, particularly a cell-mediated cytolyticresponse, against a cell expressing an antigen against which thebiotinylated components are directed. The fusion protein may simplyenhance the immune response (thus serving as an immunogeniccomposition), or confer protective immunity (thus serving as a vaccine).

Thus, the heat shock protein fusion and biotinylated components producedas described above may be purified to a suitable purity for use as apharmaceutical composition. Generally, purified compositions will haveone species that comprises more than about 85 percent of all speciespresent in the composition, more than about 85%, 90%, 95%, 99% or moreof all species present. The object species may be purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single species. A skilled artisan may purify a heatshock protein fusion and biotinylated components, or a non-covalentcomplex of the same, using standard techniques for purification, forexample, immunoaffinity chromotography, size exclusion chromatography,etc. in light of the teachings herein. Purity of a protein may bedetermined by a number of methods known to those of skill in the art,including for example, amino-terminal amino acid sequence analysis, gelelectrophoresis and mass-spectrometry analysis.

Accordingly, provided are pharmaceutical compositions comprising theabove-described heat shock protein fusion and biotinylated components,or a non-covalent complex of the same. In one aspect, provided arepharmaceutically acceptable compositions which comprise atherapeutically-effective amount of one or more of the pharmaceuticalcompositions described herein, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Inanother aspect, in certain embodiments, the pharmaceutical compositionsmay be administered as such or in admixtures with pharmaceuticallyacceptable carriers and may also be administered in conjunction withother agents. Conjunctive (combination) therapy thus includessequential, simultaneous and separate, or co-administration in a waythat the therapeutic effects of the first administered one has notentirely disappeared when the subsequent is administered.

The heat shock protein fusion and biotinylated components, or anon-covalent complex of the same, as described herein can beadministered to a subject in a variety of ways. The routes ofadministration include systemic, peripheral, parenteral, enteral,topical, and transdermal (e.g., slow release polymers). Any otherconvenient route of administration can be used, for example, infusion orbolus injection, or absorption through epithelial or mucocutaneouslinings. In addition, the compositions described herein can contain andbe administered together with or without other pharmacologicallyacceptable components such as biologically active agents (e.g.,adjuvants such as alum), surfactants (e.g., glycerides), excipients(e.g., lactose), carriers, diluents and vehicles. Furthermore, thecompositions can be used ex vivo as a means of stimulating white bloodcells obtained from a subject to elicit, expand and propagateantigen-specific immune cells in vitro that are subsequentlyreintroduced into the subject.

Further, a heat shock protein fusion protein can be administered by invivo expression of a nucleic acid encoding such protein sequences into ahuman subject. Expression of such a nucleic acid and contact withbiotinylated components can also be achieved ex vivo as a means ofstimulating white blood cells obtained from a subject to elicit, expandand propagate antigen-specific immune cells in vitro that aresubsequently reintroduced into the subject. Expression vectors suitablefor directing the expression of heat shock protein fusion proteins canbe selected from the large variety of vectors currently used in thefield. Preferred will be vectors that are capable of producing highlevels of expression as well as are effective in transducing a gene ofinterest. For example, recombinant adenovirus vector pJM17 (All et al.,Gene Therapy 1:367-84 (1994); Berkner K. L., Biotechniques 6:616-241988), second generation adenovirus vectors DE1/DE4 (Wang and Finer,Nature Medicine 2:714-6 (1996)), or adeno-associated viral vectorAAV/Neo (Muro-Cacho et al., J. Immunotherapy 11:231-7 (1992)) can beused. Furthermore, recombinant retroviral vectors MFG (Jaffee et al.,Cancer Res. 53:2221-6 (1993)) or LN, LNSX, LNCX, LXSN (Miller andRosman, Biotechniques 7:980-9 (1989)) can be employed. Herpes simplexvirus-based vectors such as pHSV1 (Geller et al., Proc. Nat'l Acad. Sci.87:8950-4 (1990) or vaccinia viral vectors such as MVA (Sutter and Moss.Proc. Nat'l Acad. Sci. 89:10847-51 (1992)) can serve as alternatives.

Frequently used specific expression units including promoter and 3′sequences are those found in plasmid cDNA3 (Invitrogen), plasmid AH5,pRC/CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927(1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSRa(DNAX, Palo Alto, Calif.). The introduction of genes into expressionunits and/or vectors can be accomplished using genetic engineeringtechniques, as described in manuals like Molecular Cloning and CurrentProtocols in Molecular Biology (Sambrook, J., et al., Molecular Cloning,Cold Spring Harbor Press (1989); Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience (1989)). A resulting expressible nucleic acid can beintroduced into cells of a human subject by any method capable ofplacing the nucleic acid into cells in an expressible form, for exampleas part of a viral vector such as described above, as naked plasmid orother DNA, or encapsulated in targeted liposomes or in erythrocyteghosts (Friedman, T., Science, 244:1275-1281 (1989); Rabinovich, N. R.et al., Science. 265:1401-1404 (1994)). Methods of transduction includedirect injection into tissues and tumors, liposomal transfection (Fraleyet al., Nature 370:111-117 (1980)), receptor-mediated endocytosis(Zatloukal et al., Ann. N.Y. Acad. Sci. 660:136-153 (1992)), andparticle bombardment-mediated gene transfer (Eisenbraun et al., DNA &Cell. Biol. 12:791-797 (1993)).

The amount of heat shock protein fusion and biotinylated components, ora non-covalent complex of the same, in the compositions of the presentinvention is an amount which produces an effective immunostimulatoryresponse in a subject. An effective amount is an amount such that whenadministered, it induces an immune response. In addition, the amount ofheat shock protein fusion and biotinylated components, or a non-covalentcomplex of the same, administered to the subject will vary depending ona variety of factors, including the heat shock protein fusion andbiotinylated component employed, the size, age, body weight, generalhealth, sex, and diet of the subject as well as on its generalimmunological responsiveness. Adjustment and manipulation of establisheddose ranges are well within the ability of those skilled in the art. Forexample, the amount of heat shock protein fusion, biotinylatedcomponents, or a non-covalent complex of the sam, ecan be from about 1microgram to about 1 gram, preferably from about 100 microgram to about1 gram, and from about 1 milligram to about 1 gram. An effective amountof a composition comprising an expression vector is an amount such thatwhen administered, it induces an immune response against the antigenagainst which the pharmaceutical composition is directed. Furthermore,the amount of expression vector administered to the subject will varydepending on a variety of factors, including the heat shock proteinfusion expressed, the size, age, body weight, general health, sex, anddiet of the subject, as well as on its general immunologicalresponsiveness. Additional factors that need to be considered are theroute of application and the type of vector used. For example, whenprophylactic or therapeutic treatment is carried out with a viral vectorcontaining a nucleic acid encoding heat shock protein fusion, theeffective amount will be in the range of 10⁴ to 10¹² helper-free,replication-defective virus per kg body weight, preferably in the rangeof 10⁵ to 10¹¹ virus per kg body weight and most preferably in the rangeof 10⁶ to 10¹⁰ virus per kg body weight.

Determination of an effective amount of fusion protein and biotinylatedcomponents, or a non-covalent complex of the same, for inducing animmune response in a subject is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immune response using techniques that are well known inthe art. One having ordinary skill in the art could readily optimizeadministration to humans based on animal data. Dosage amount andinterval may be adjusted individually. For example, when used as avaccine, the proteins and/or strains of the invention may beadministered in about 1 to 3 doses for a 1-36 week period. Preferably, 3doses are administered, at intervals of about 3-4 months, and boostervaccinations may be given periodically thereafter. Alternate protocolsmay be appropriate for individual patients. A suitable dose is an amountof protein or strain that, when administered as described above, iscapable of raising an immune response in an immunized patient sufficientto protect the patient from the condition or infection for at least 1-2years.

The compositions may also include adjuvants to enhance immune responses.In addition, such proteins may be further suspended in an oil emulsionto cause a slower release of the proteins in vivo upon injection. Theoptimal ratios of each component in the formulation may be determined bytechniques well known to those skilled in the art.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to enhance the immune response. Most adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a specific or nonspecificstimulator of immune responses, such as lipid A, or Bortadellapertussis. Suitable adjuvants are commercially available and include,for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant(Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc.,Rahway, N.J.). Other suitable adjuvants include alum, biodegradablemicrospheres, monophosphoryl lipid A, quil A, SBAS1c, SBAS2 (Ling etal., 1997, Vaccine 15:1562-1567), SBAS7, Al(OH)₃ and CpG oligonucleotide(WO96/02555).

In the vaccines of the present invention, the adjuvant may induce a Th1type immune response. Suitable adjuvant systems include, for example, acombination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) together with an aluminum salt. Anenhanced system involves the combination of a monophosphoryl lipid A anda saponin derivative, particularly the combination of 3D-MLP and thesaponin QS21 as disclosed in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol as disclosed inWO 96/33739. Previous experiments have demonstrated a clear synergisticeffect of combinations of 3D-MLP and QS21 in the induction of bothhumoral and Th1 type cellular immune responses. A particularly potentadjuvant formation involving QS21, 3D-MLP and tocopherol in anoil-in-water emulsion is described in WO 95/17210 and may comprise aformulation.

Kits

The present invention provides kits for expressing or administering aheat shock protein fused to a biotin-binding protein. Such kits may becomprised of nucleic acids encoding heat shock protein fused to abiotin-binding protein. The nucleic acids may be included in a plasmidor a vector, e.g., a bacterial plasmid or viral vector. Other kitscomprise a heat shock protein fused to a biotin-binding protein.Furthermore, the present invention provides kits for producing and/orpurifying a heat shock protein fused to a biotin-binding protein. Suchkits may optionally include biotinylated components or biotinylationreagents as described herein.

The present invention provides kits for preventing or treatinginfectious or malignant disease in a patient. For example, a kit maycomprise one or more pharmaceutical compositions as described above andoptionally instructions for their use. In still other embodiments, theinvention provides kits comprising one more pharmaceutical compositionand one or more devices for accomplishing administration of suchcompositions.

Kit components may be packaged for either manual or partially or whollyautomated practice of the foregoing methods. In other embodimentsinvolving kits, instructions for their use may be provided.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Example 1

i) Production of MTBhsp70

MTBhsp70 was subcloned into the expression vector pET45b(+) by firstmodifying the vector to introduce the desired restriction site SfiI.This modification allows introduction of the MTBhsp70 protein at theNotI/XhoI site and other proteins such as scFvs, antigens, Avidin, etc.at the SfiI/NotI site (FIG. 1). This particular approach can be modifiedto introduce the desired proteins at the C-terminal of MTBhsp70. Usingthe MTBhsp70 plasmid provided by Dr. Richard Young, restriction sitesNotI and XhoI were introduced at the 5′ and 3′ end respectively asdescribed in FIG. 2.

Digestion of the amplified MTBhsp70 fragment shown in lane 1 of FIG. 2with the restriction enzymes NotI and XhoI unexpectedly revealed 2 bands(FIG. 3). Sequencing analyses revealed that MTBhsp70 contains internalNotI and SfiI restriction sites. These were removed using the strategydepicted in FIG. 4. The resulting MTBhsp70 pET-45b(+) construct was thenused to transform competent BL21(DE3) bacteria. Expression of MTBhsp70was induced by adding 1 mM IPTG. Cells were grown in LB medium at 37° C.to an OD₆₀₀ of 0.5. Cells were spun down and suspended in LB mediumcontaining 1 mM IPTG and growth continued for 4 hours at the indicatedtemperature. Cells were fractionated and aliquots were run on SDS-PAGEand proteins were stained with Coomassie Blue. When induced cells weregrown at 37° C. the majority of the MTBhsp70 protein was found ininsoluble inclusion bodies. By reducing the growth temperature postinduction to 30° C., large amounts of soluble MTBhsp70 were produced.The MTBhsp70 protein found in the soluble and periplasmic fractions ofBL21(DE3) grown at 30° C. was successfully purified by metal affinitychromatography (MAC) using Cobalt spin columns. Cells were grown at 37°C. to an OD₆₀₀ of 0.5 and then spun down. Cells were suspended in growthmedia containing 1 mM IPTG and allowed to grow for 4 hours at 30° C.Cells were fractionated according to standard methods consisting ofsolubilization with B-PER reagent from Pierce.

ii) Production of MTBhsp70-Fusion Proteins

In order to demonstrate the immunostimulatory properties ofMTBhsp70-fusion proteins, an Ovalbumin peptide-MTBhsp70 and twoscFv-MTBhsp70 fusion products were constructed.

-   -   a. Ova-257-264-MTBhsp70 fusion protein. Dr. Young's group        established that the Ovalbumin's immunodominant peptide consists        of residues 257-264 (SIINFEKL) (SEQ ID NO: 4). This peptide was        fused to the N-terminal region of MTBhsp70 by digesting the        MTBhsp70 pET-45b(+) plasmid with SfiI and NotI and introducing a        linker coding for the immunodominant peptide that is also        digested with SfiI and NotI (FIG. 5). Upon ligation, a number of        colonies were obtained, and their identities were confirmed by        sequencing (FIG. 6). As observed with MTBhsp70, induction of        Ova-257-264-MTBhsp70 is optimum when the growth temperature,        post-IPTG induction, is kept at 30° C. Successful expression of        Ova254-264-MTBhsp70 was obtained in the soluble fraction of        BL21(DE3).    -   b. scFv-MTBhsp70 fusion proteins. scFvs were fused to the        N-terminal of MTBhsp70. A human combinatorial scFv phage display        library was constructed and used it to select Ovalbumin specific        scFvs. The other scFv, MOV18, is specific for the high affinity        Folate Receptor expressed on ovarian cancer cells. The cloning        method is similar to the approach used for the introduction of        the Ova₂₅₄₋₂₆₄ peptide at the N-terminal of MTBhsp70. The        SfiI/NotI scFv portion was purified from their respective        plasmids followed by ligation into the SfiI/NotI digested        expression vector MTBhsp70 pET-45b(+). The anti-Ovalbumin scFvs        had several non-sense mutations that had to be removed by site        directed mutagenesis. However, upon induction of bacteria        carrying both constructs, it was found that induction with IPTG        resulted in the fusion proteins being expressed in inclusion        bodies.

iii) Production of Avidin-Linker-MTBhsp70

A fusion of Avidin to a linker element and to MTBhsp70 may be used forthe production of a self-assembling vaccine. This is illustrated in FIG.7 where the linker portion is illustrated as a line between Avidin andheat shock proteins. Avidin is a homo tetrameric glycosylated proteinwith a molecular weight of 68,000 (thus each subunit is 17,000 dalton).The wild-type (tetrameric) or the monomeric form described by Dr. MarkkuS. Kulomaa may be produced and used as provided herein. These moleculesare described according to the scheme of FIG. 8. The monomeric form ofavidin differs from the wild-type at 5 amino acid positions. This isillustrated in FIG. 9. A series of primers and linkers were used toassemble each Avidin construct.

Minor mutations were corrected by PCR based mutagenesis. This isillustrated in FIG. 10, which shows two changes that were made tomonomeric avidin (clone M1). Both monomeric and wild-typeAvidin-linker-MTBhsp70 constructs were successfully obtained as shown inFIG. 11. Both constructs lead to abundant protein production uponinduction with IPTG in E. coli BL21(DE3). However, the inducedAvidin-linker-MTBhsp70 proteins are found mostly in inclusion bodies,which may be solubilized, denatured and refolded.

Following denaturation with guadinium hydrochloride and DTT, theproteins are diluted in 4 different refolding buffers (1) Tween 40,Cystine; 2) Tween 60, Cystine; 3) CTAB, Cystine; 4) SB3-14, Cystine)containing cystine to quench excess DTT. After addition of 3% CAsolution the proteins are allowed to refold overnight at roomtemperature. To assess the level of refolding, refolded protein aliquotswere added to biotin-coated wells. Thus, properly refoldedAvidin-Linker-MTBhsp70 proteins would bind tightly to the bottom ofthose wells. The amount of plate-bound proteins were determined by usinga monoclonal antibody against MTBhsp70 followed by the detection ofbound anti-MTBhsp70 using Horse Radish Peroxidase (HRP) labeled Goatanti mouse (H+L) antibodies. The optical density was recorded at 450 nmand expressed the results as percent maximum signal. The results aredepicted in FIG. 12. Proper refolding of each protein may includesubjected each protein to different conditions. In order to assess thebiological activity of the refolded MTBhsp70 portion of these proteins,their ability to hydrolyze ATP may be measured. Furthermore, chemotacticeffects of MTBhsp70 on THP-1 cells may be determined.

Example 2

The self-assembly and biological activities ofm/wAvidin-Linker-N-MTBhsp70 proteins may be determined as follows:

-   1) Determination of ATPase activity. The N-terminal portion of    MTBhsp70 is known to hydrolyze ATP under certain assay conditions.    The proper refolding of our m/wAvidin-Linker-N-MTBhsp70 proteins may    be assessed by comparing their ATPase activity to that of a    commercial preparation and our own MTBhsp70.-   2) Determination of chemotactic activity/induction of chemokine    production. Another assay of proper refolding of the MTBhsp70    portion of our m/wAvidin-Linker-N-MTBhsp70 proteins is their    successful induction of CC chemokines from the cell line THP-1.    Exposure of the monocyte cell line THP-1 to MTBHhsp70 is known to    induce the production of RANTES, MIP-1α and MIP-1β. In turn these    chemokines should stimulate chemotactic activity.-   3) Self-assembly. Refolded m/wAvidin-Linker-N-MTBhsp70 proteins bind    biotin. To demonstrate self-assembly, the ability of these proteins    to form a stable complex with biotinylated molecules may be    demonstrated. Binding to biotinylated Ovalbumin and biotinylated    eGFP may be demonstrated. Successful assembly is assessed by    immunoprecipitation with anti-MTBhsp70 and anti-biotinylated    antigen.-   4) In vivo induction of immunity. The immunostimulatory activity of    the self-assembled vaccines may be demonstrated by measuring the    activation of CD8 T-cells upon immunization of C57BL/6 mice with    biotinylated Ovalbumin-m/wAvidin-Linker-N-MTBhsp70 and biotinylated    Ova₂₅₇₋₂₆₄-m/wAvidin-Linker-N-MTBhsp70.

Example 3

An MTBHSP70 Construct with Avidin Self Assembled with Biotinylated HRPand Biotinylated Anti-OVA Antibodies

Methods

i) Plasmid Constructs

MTBHSP70 was subcloned into the expression vector pET45b(+) and thenlinked the CD8 specific Ovalbumin peptide_((Ova257-264)) at theN-terminal. Avidin and monomeric avidin based on published sequences wasassembled and linked at the N-terminal of MTBHSP70 into the expressionvector pET45b(+).

ii) Protein Expression and Purification

E. coli BL21(DE3) was transformed with the various constructs andexpression was induced by addition of IPTG. Proteins were purified byIMAC in the presence of TritonX-114 (Sigma) to remove endotoxins.

iii) Immunizations

C57BL/6 male mice were immunized with Ovalbumin or theOva₂₅₇₋₂₆₄-L-MTBHSP70 subcutaneously and sacrificed at day 30.

iv) Outcome Measures

-   -   a. Interferon-γ. After 2 subcutaneous immunizations, mice were        sacrificed at day 30 and splenocytes prepared. Splenocytes        (2×106 per well) were incubated at 37° C. for 4 hours in        presence of brefeldin A (golgi plug) and Ova₂₅₇₋₂₆₄ peptide or        an irrelevant peptide. The incubation was stopped by washing        cells with 5% FBS in PBS followed by staining for CD3, CD4, CD8        and Interferon-γ after cells were permeabilized with BD's        Cytofix/Cytoperm solution. Cell staining was evaluated by flow        cytometry and analyzed using FlowJo.    -   b. Pentamer staining. Cells were treated as described above with        the addition that they were initially treated with R-PE        conjugated recombinant murine MHC Pentamer H-2Kb SIINFEKL (SEQ        ID NO: 4)(from PROIMMUNE).    -   c. Self-Assembly. An ELISA based assay was employed. Various        concentrations of biotinylated Horse Radish Peroxidase (HRP) and        purified and refolded avidin-MTBHSP 70 were mixed. The mixture        was added to His-Grab plates (ThermoScientific) and unbound        biotinylated HRP removed by washing with PBS 0.1% Tween 20        followed by addition of TMB. The reaction was stopped after 20        minutes and the results read at 450 nm.        Results

6×His-MTBHSP 70 (“6×His” disclosed as SEQ ID NO:5) and6×His-Ova(257-264)-MTBHSP 70 fusion proteins (“6×His” disclosed as SEQID NO:5) were expressed in E. coli BL21(DE3) cells. In each case,NotI-XhoI MTBHsp70 fragments were used in the fusion constructs. Proteinexpression was induced by addition of IPTG. Cells were lysed andfractionated into soluble (#1 and #4), periplasmic (#2 and #5) andinclusion body fractions. Aliquots from each fractions were subjected todenaturing SDS-PAGE on 4-12% Bis-Tris NUPAGE gels (Invitrogen).

i) Expression of Avidin and Monomeric Avidin Linked to MTBHSP70 in E.coli

Wild-type Avidin (wAvidin) and monomeric Avidin were assembled andcloned into pET45b(+). Fusion constructs were prepared consisting of6×His-Avidin (“6×His” disclosed as SEQ ID NO:5) linked at the N-terminalof MTBHSP 70. Each plasmid construct was used to transform E. coliBL21(DE3) and expression induced with IPTG.

ii) Immunization #1

C57BL/6 male mice were immunized subcutaneously on days 1 and 17 andsacrificed on Day 30 as follows:

-   -   Ovalbumin+CFA    -   Ovalbumin    -   Ovalbumin+MTBhsp70    -   Ova_(peptide)-MTBhsp70

iii) Outcome Measure

Production of Interferon gamma upon stimulation of splenocytes with CD8peptide SIINFEKL (SEQ ID NO: 4) (Ova₂₅₇₋₂₆₄) was measured, and theresults are shown in FIG. 16 and Table 1 below.

TABLE 1 Percent CD + 8 splenocytes producing Interferon-γ uponstimulation with Ova₂₅₇₋₂₆₄ Percent CD8⁺Ifnγ⁺ (minus unstimulated) CFA0.39 Ovalbumin 0.09 Ovalbumin + MTBhsp70 0.23 Ovapeptide − MTBhsp70 0.22

iv) Immunization #2

C57BL/6 mice (males) were immunized subcutaneously as described forimmunization #1. The immunization groups were as follows:

-   -   Group A CFA+Ovalbumin (3 mice)    -   Group B CFA+Ovapeptide (257-264) (3 mice)    -   Group C Ovapeptide-MTBhsp70 fusion (3 mice)    -   Group D Ovapeptide+MTBhsp70 (3 mice)    -   Group E MTBhsp70 (3 mice)    -   Group F saline 1 mouse

v) Outcome Measure

Splenocytes were harvested and stained with CD3, CD4, CD8 andH-2Kb/SIINFEKL (SEQ ID NO: 4) (OVA) Pentamer from Prolmmune.

vi) Conclusion

HSP fusion protein constructs were developed and expressed in E. coli.Fusion of Ovapeptide 257-264 to the N-terminal of MTBHSP 70 resulted insuccessful expansion of antigen specific T-cells as measured byInterferon-γ production and H-2Kb/SIINFEKL (SEQ ID NO: 4) (OVA)staining.

Example 4

Modified Constructs to Include Avidin at the N- or C-Terminal of MTBHSP70

i) Measuring Self-Assembly

Self-assembly of the fusion protein Avidin-MTBHSP 70 with biotinylatedhorseradish peroxidase (HRP) was assessed using His-Grab plate ELISA.Inclusion bodies were solubilized with guanidium chloride and slowlydialyzed against buffer containing decreasing concentrations ofguanidium. The partially refolded protein was incubated with differingamounts of biotinylated HRP prior to being added to a His-Grab plate.The plate was read at 450 nm.

ii) Assessing Targeting of Self-Assembled Vaccine

Targeting of self-assembled vaccine was assessed by flow cytometry. Thepartially refolded Avidin MTBHSP 70 protein was incubated withbiotinylated anti-Ovalbumin antibodies and the complex captured onmagnetic His-tag specific beads. After removal of excess antibodies,Alexa Fluor 555 labeled Ovalbumin was added. Samples were analyzed byflow cytometry in the R-PE channel. A very weak difference was observedwhen compared with beads alone. Targeted self-assembly was confirmed bystatistical analyses.

iii) Conclusion

Fusion proteins consisting of either peptide antigens or avidin at theN-term of MTBHSP 70 have been successfully expressed. Immunization withOvapeptide (257-264) fused in frame at the N-terminal of MTBHSP 70 ledto the induction of an antigen specific immune response as measured byInterferon gamma production and the expansion of CD8+ T-cells (pentamerstaining) Constructs expressing avidin at the N- or C-terminal of MTBHSP70 were also designed, thus allowing self assembly of this constructwith biotinylated clinically relevant antibodies. The avidin fusionproteins were successfully expressed in E. coli but were found ininclusion bodies. Refolding of these proteins was partially successfuland is currently being optimized. Low efficiency self assembly ofMTBHSP70-avidin with biotinylated monoclonal antibody was demonstratedin preliminary experiments.

References

-   Chen, W., U. Syldath, et al. (1999). “Human 60-kDa heat-shock    protein: a danger signal to the innate immune system.” J Immunol    162(6): 3212-9.-   Laitinen, O. H., H. R. Nordlund, et al. (2007). “Brave new    (strept)avidins in biotechnology.” Trends Biotechnol 25(6): 269-77.-   Nordlund, H. R., V. P. Hytonen, et al. (2005). “Tetravalent    single-chain avidin: from subunits to protein domains via circularly    permuted avidins.” Biochem J 392(Pt 3): 485-91.-   Srivastava, P. K. and R. G. Maki (1991). “Stress-induced proteins in    immune response to cancer.” Curr Top Microbiol Immunol 167: 109-23.-   Zugel, U. and S. H. Kaufmann (1999). “Role of heat shock proteins in    protection from and pathogenesis of infectious diseases.” Clin    Microbiol Rev 12(1): 19-39.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The appended claims are notintended to claim all such embodiments and variations, and the fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

REFERENCES

Related approaches are disclosed in PCT/US2007/061554 by the presentinventors, of which the entire contents are incorporated herein byreference. Incorporated by reference in their entirety also are anypolynucleotide and protein sequences which reference an accession numbercorrelating to an entry in the public database of the National Centerfor Biotechnology Information (NCBI) on the world wide web atncbi.nlm.nih.gov. The contents of all cited references includingliterature references, issued patents, published or non published patentapplications as cited throughout this application are also herebyexpressly incorporated by reference.

The invention claimed is:
 1. A pharmaceutical composition comprising: apharmaceutically acceptable carrier and a heat shock protein fused to abiotin-binding protein, wherein the biotin-binding protein isnon-covalently bound to a biotinylated engineered antibody, thebiotinylated engineered antibody is a biotin-containing recombinantmolecule that comprises at least an antibody fragment, and the antibodyfragment comprises an antigen binding site.
 2. The pharmaceuticalcomposition of claim 1, wherein the biotin-binding protein is selectedfrom the group consisting of avidin, streptavidin, and neutravidin. 3.The pharmaceutical composition of claim 1, wherein the heat shockprotein is a mammalian heat shock protein or a bacterial heat shockprotein.
 4. The pharmaceutical composition of claim 1, wherein the heatshock protein is selected from the group consisting of an MTBhsp70 heatshock protein and a human heat shock protein.
 5. The pharmaceuticalcomposition of claim 1, which is a vaccine.
 6. A method for producing aself-assembling pharmaceutical composition of claim 1, comprisingcontacting a heat shock protein fused to a biotin-binding protein,wherein the biotin-binding protein is non-covalently bound to abiotinylated engineered antibody, sufficient to form a non-covalentcomplex of the heat shock protein and the biotinylated engineeredantibody, the biotinylated engineered antibody is a biotin-containingrecombinant molecule that comprises at least an antibody fragment, andthe antibody fragment comprises an antigen binding site.
 7. Thepharmaceutical composition of claim 1, wherein the biotinylatedengineered antibody comprises a scFv fragment.
 8. The pharmaceuticalcomposition of claim 1, wherein the biotinylated engineered antibodycomprises a Fab fragment.
 9. The pharmaceutical composition of claim 1,wherein the biotinylated engineered antibody is multivalent.
 10. Thepharmaceutical composition of claim 1, wherein the biotinylatedengineered antibody is tetravalent.
 11. A method for inducing an immuneresponse in a subject, comprising administering to the subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a heat shock protein fused to a biotin-binding protein,wherein the biotin-binding protein is non-covalently bound to abiotinylated engineered antibody, the biotinylated engineered antibodyis a biotin-containing recombinant molecule that comprises at least anantibody fragment, and the antibody fragment comprises an antigenbinding site.
 12. The method of claim 11, wherein the biotin-bindingprotein is selected from the group consisting of avidin, streptavidin,and neutravidin.
 13. The method of claim 11, wherein the heat shockprotein is a mammalian heat shock protein or a bacterial heat shockprotein.
 14. The method of claim 11, wherein the heat shock protein isselected from the group consisting of an MTBhsp70 heat shock protein anda human heat shock protein.
 15. The method of claim 11, wherein thepharmaceutical composition is administered to the subject as anon-covalent complex.